Abstract

All organisms have devised strategies to counteract energy depletion and promote fitness for survival. We show here that cellular energy depletion puts into play a surprising strategy that leads to absorption of exogenous fuel for energy repletion. The energy-depletion-sensing kinase AMPK binds, phosphorylates, and activates the transcriptional coactivator SRC-2, which in a liver-specific manner promotes absorption of dietary fat from the gut. Hepatocyte-specific deletion of SRC-2 results in intestinal fat malabsorption and attenuated entry of fat into the blood stream. This defect can be attributed to AMPK- and SRC-2-mediated transcriptional regulation of hepatic bile acid (BA) secretion into the gut, as it can be completely rescued by replenishing intestinal BA or by genetically restoring the levels of hepatic bile salt export pump (BSEP). Our results position the hepatic AMPK-SRC-2 axis as an energy rheostat, which upon cellular energy depletion resets whole-body energy by promoting absorption of dietary fuel.

A) Expression of various BA transporter genes was measured via relative quantitation by QPCR in primary hepatocytes exposed to DMSO or to CDCA from WT and SRC-2 LKO mice. Statistical comparison was performed between WT and SRC-2 KO PHs in each treatment group.B) HepG2 liver hepatoma cells were transfected with a reporter-gene plasmid driven by the wild-type mouse BSEP promoter and the same promoter with a mutated FXRE motif, together with expression plasmids for SRC-2 and FXR and exposed to CDCA. Reporter-gene levels were determined 48 hours after transfection. The empty vector (EV) value was fixed at 1 and the rest of the values are compared relative to that.C) HepG2 liver hepatoma cells were transfected with a reporter-gene plasmid driven by the wild-type mouse BSEP promoter together with expression plasmids for WT SRC-2, SRC-2 mutant with AD1 deletion, SRC-2 mutant with AD2 deletion and SRC-2 mutant with both AD1 and AD2 deletions, and FXR, and exposed to CDCA. Reporter-gene levels were determined 48 hours after transfection. The empty vector (EV) value was fixed at 1 and the rest of the values are compared relative to that.D) In vivo ChIP assays were performed using liver tissue from WT mice with 150-200 bp amplicons flanking the region containing the FXRE motif of the BSEP promoter and an irrelevant region 3000 bp upstream of the transcription start site. Sybr-Green QPCR (normalized to input) was used to assess SRC-2 occupancy of the BSEP promoter, using two different antibodies that targeted different regions of SRC-2. Control antibody recognized mouse IgG.E) In vivo ChIP assays were performed using liver tissue from WT and FXR knockout mice with primers flanking the region containing the FXRE motif of the BSEP promoter. Sybr-Green QPCR (normalized to input) was used to assess SRC-2 occupancy of the BSEP promoter, using an SRC-2 specific antibody. Control antibody recognized mouse IgG.Data are represented as mean + SEM. For all gene expression data unpaired student's t-test was used for evaluation of statistical significance. One asterisk indicates p < 0.05, two asterisks p < 0.01 and three asterisks p < 0.001.See also

Hepatic AMPK increases the intrinsic transcriptional activity of SRC-2, and drives it to the BSEP promoter

A) HepG2 liver hepatoma cells were transfected with pG5Luc (5 Gal4 binding sites driving luciferase expression) together with pBIND SRC-2 (SRC-2:Gal4 DNA binding domain fusion protein) and exposed to 1 mM AICAR. Luciferase levels were determined 48 hours after transfection. The vehicle value was fixed at 1 and the rest of the values are compared relative to that.B) HepG2 liver hepatoma cells were transfected with pG5Luc together with pBIND SRC-2 and either empty vector (EV) or dominant negative AMPK alpha 2 (DnAMPK) and exposed to 0.3 mM, 0.6 mM and 1 mM AICAR. Luciferase levels were determined 48 hours after transfection. The vehicle value was fixed at 1 and the rest of the values are compared relative to that.C) HepG2 cell were treated with 1 mM AICAR for 30 min and subjected to immunoprecipitation with an AMPKa2 specific antibody. Immunoprecipated samples were subjected to immunoblotting along with 10% input as indicated.D) Purified full length SRC-2 protein was subjected to in vitro phosphorylation with or without purified AMPK holoenzyme and AMP as indicated.E-F) Hepatic BSEP expression and BA content was measured via relative quantitation by QPCR in the liver of WT and AMPK alpha1/alpha2 double knockout (AMPK alpha DKO) mice that were fasted for 24 hours (n = 7-8 mice per group).G) Hepatic BSEP expression was measured via relative quantitation by QPCR in the liver of mice that were exposed to either PBS or 0.5 mg/g BW AICAR for 12 hours and fed standard chow (n = 6 mice per group).H) BSEP expression was measured via relative quantitation by QPCR in primary hepatocytes from WT and SRC-2 LKO mice exposed to 1 mM AICAR for 18 hours.I-J) ChIP assays were performed using HepG2 cells exposed to either vehicle or 1 mM AICAR for 30 min. with primers flanking the region containing the FXRE motif of the BSEP promoter. Sybr-Green QPCR (normalized to input) was used to assess SRC-2 or AMPK alpha 2 occupancy of the BSEP promoter upon chromatin immunoprecipitation, using an SRC-2 or AMPK a2 specific antibody. Control antibody recognized mouse IgG.All gene expression data are represented as mean + SEM. Unpaired student's t-test was used for evaluation of statistical significance. One asterisk indicates p < 0.05, two asterisks p < 0.01 and three asterisks p < 0.001.See also